Detection of unique target sequences in the complexity of the human genome requires highly specific detection reagents. This form of gene detection permits target sequence variants to be screened for and it represents one of the most important modalities of gene analysis. The hybridization of single oligonucleotides in general does not afford the requisit specificity, while the combination of two probes such as in PCR (polymerase chain reaction) or OLA (oligonucleotide ligation assay) allows specific detection of unique target sequences.
In situations where many different target sequences are investigated together in a sample by techniques such as PCR or OLA, the risk of “crosstalk” between noncognate pairs of probes, giving rise to false detection signals, increases exponentially with multiplexing.
WO 95/22623 discloses a probe designed to be circularized in the presence of a target sequence and caused to close around the target-containing nucleic acid strand such that the cyclic probe will interlock with and thereby be efficiently linked to the target nucleic acid to be detected. Because of the helical nature of double-stranded nucleic acids, such as DNA, circularized probes will be wound around the target strand, topologically connecting probes to target molecules through catenation, in a manner similar to a padlock. Such covalent catenation of probe molecules to target sequences results in the formation of a hybrid that resists extreme washing conditions, serving to reduce non-specific signals in genetic assays. Any probes hybridizing in a non-specific manner may therefore be removed by subjecting the target to non-hybridizing conditions and/or exonuclease activity. Provided that the circular probe is properly detectable, such as by being suitably labelled, it may then be detected to indicate the presence of the target molecule.
In an assay based on such circularizable probes only intramolecular reactions give rise to a signal, thereby greatly improving the potential for multiplexing.